This invention relates to microbiological test processes and apparatus, and especially to microbial identification testing and to testing for minimum inhibitory concentrations of antibiotics in relation to clinical isolates of pathogenic microorganisms (M.I.C.testing).
It has long been the clinical laboratory practice to test microbes isolated clinically from patients and other sources for their sensitivity to antimicrobial materials and for their metabolic growth requirements. The aim of such tests is to identify the microbes and/or assess their sensitivity to antimicrobial agents in the clinical context.
Numerous methods and apparatus have been developed to aid these tests. Particularly convenient for this purpose are prepared microtitre plates provided with a series of culture wells, each containing a graduated small quantity either of an antimicrobial agent, such as for example an antibiotic, and/or a growth-promoting material, such as a vitamin, sometimes together with other ingredients for a microbial culture medium, and often in dried and otherwise stabilised form. Examples of such microtitre plates and their use are given by U.S. Pat. No. 3,713,985 (Astle), French Patent No. 1,488,866 (Sclavo), and GB Patent Specification No 1 574 707 (Unilever). The latter specification describes the production of a particularly well-stabilised form of microtitre plate.
In use, plates of the above kinds, and other suitable devices, are inoculated with all the necessary culture materials and inoculum, and incubated until the culture result appears. Often, this culture result is seen as a "button" of growth at the bottom of a microtitre well, and typically requires at least 18 hours of incubation. (With certain fast-growing micro-organisms, this method can give "button" formation as early as 7 hours but at this stage it is generally not possible to distinguish a sensitivity test result.)
It is desirable to reduce the incubation time required before a culture result can be detected.
Certain proposals to this end have already been made. Particular attempts are described for example in U.S. Pat. No. 4,236,211 (Pfizer) (measurement of light scattering by micro-organisms in a limited number of cultures after about 5 hours, see also U.S. Pat. No. 3,832,532 and GB Patent No. 1,554,134 (Pfizer). The use of these methods calls for complex and expensive apparatus and/or extensive calculations based on the light-scattering observed. U.S. Pat. No. 4,242,447 (Bio Research) particularly describes estimation of the bacterial content of liquids by the use of fluorescence detection after exposure of bacterially contaminated specimen liquids to an enzyme-inducing medium for about 15-30 minutes, followed by reaction of the mixture with fluorescein di-beta-galactoside and fluorimetry.
U.S. Pat. No. 3,509,026 (Litton Systems Inc.) is especially relevant because it describes a culture procedure for indicating antibiotic sensitivity of microorganisms, involving the use of inert pads on a mounting strip, each containing a culture medium, antibiotic, and flavone-3-diphosphate as a hydrolysable substrate to indicate bacterial growth by fluorescence. It is suggested in U.S. Pat. No. 3,509,026 that the system described will allow rapid assessment of antibiotic sensitivity, and examples are given showing the results of examination of the cultures under fluorescent light after 4 hours' culture. Although some indications of antibiotic sensitivities were obtained after 4 hours' culture, there are several results denoted as unclear among the examples, and it appears to us to be at least difficult to prepare a simple and reliable system based on this technique.
More recently, T. Urban and C. Jarstrand have described in J. Antimicrobial Chemotherapy (1981) 8, 363-369 a bacterial identification and MIC method based on commercially available Sensititre test plates, with the addition after 4 hours' culture of nitroblue tetrazolium and phenazine methosulphate to give blue formazan colour where bacterial growth has occurred. NBT and PMS are however toxic to the cultures, and it is often desired to retain the cultures after MIC or identification work for further testing.
The prior art includes a number of fluorogenic substrates for enzymes of diverse origin which are known, commercially available, and have been used in enzymological procedures. Among these are a variety of fluorogenic 4-methyl-umbelliferyl derivatives (hydrolysable to 4-methyl-umbelliferone) and derivatives of 7-amino-4-methyl-coumarin, e.g. GB Patent No. 1,547,747 and European Patent No. 0,000,063 (Ajinomoto).